Project Summary The overarching goal of this training award is to acquire the skills and experience necessary to become an independent physician scientist with research expertise on fundamental mechanisms of bone marrow failure, with a final goal of developing novel therapies. Maintaining genome integrity is an essential task for cells that need to pass correct genetic information to their progeny. Cells are equipped with a variety of mechanisms to protect their genome including robust DNA repair. Hematopoietic stem cells are especially vulnerable to DNA damage and rely on Fanconi anemia DNA repair pathway that removes DNA interstrand crosslinks. Patients lacking the Fanconi anemia proteins invariably develop bone marrow failures. Unfortunately, there is no therapy that prevents bone marrow failures in Fanconi anemia. While bone marrow failure in Fanconi anemia is thought to be caused by the inappropriate repair of the DNA interstrand crosslinks, the source of that DNA damage is poorly understood. Recent studies have implicated endogenous metabolic by-products, such as acetaldehyde and formaldehyde, in acceleration of disease progression in Fanconi anemia. To identify other sources of DNA damage, we have performed a CRISPR-Cas9 screen and identified a novel source of endogenous DNA damage that needs Fanconi anemia pathway for its proper repair. In this application, we propose to validate our impactful in vitro findings in the mouse hematopoietic stem cells using in vivo transplantation assays and genetically modified mouse models. We will also assess the type of DNA damage and subsequent cellular consequences, paying special attention to the genomic instability that is caused by this endogenous source of DNA damage. We will also test whether any other protective pathways play a role in genome maintenance of hematopoietic stem cells using an unbiased in vivo CRISPR screen approach. Knowledge gained through our studies may unveil novel therapeutic targets useful for prevention of bone marrow failures in Fanconi anemia and in the general population.